There is considerable interest in the development of macromolecular chemical systems with well-defined structural properties for use as molecular scaffolding to orient and juxtapose other molecules. The motivations for pursuing these constructions include the formation of macromolecular `zeolite` lattices to enable diffraction analysis of complex guest molecules that are not readily crystallized (Seeman, N. C., In: Biomolecular Stereodynamics, ed. by R. H. Sarma, Academic Press, New York, (1981) pp. 269-277; Seeman, N. C., J. Theor., Biol., 99: 237-247 (1982); Seeman, N. C., J. Biomol., Str. & Dyns., 3: 11-34 (1985)); the caging of active biological macromolecules to form new multi- functional enzymes ( Chen, et al., J. Am. Chem Soc., 111: 6402-6407 (1989)); drug delivery systems for therapeutic macromolecules (Seeman, DNA and Cell Biology, 10: 475-486 (1991)); mechanical control on the nanometer scale (Seeman, In NANOCON Proceedings, ed. by J. Lewis et al., Nanocon, Bellevue Wash., pp. 101-123 (1989)), and the assembly of molecular electronic components (Robinson, et al., Prot. Eng., 1: 295-300 (1987); Hopfield, et al., Science. 241: 817-820 (1988)).
One of the major goals of both biotechnology and nanotechnology is the assembly of novel biomaterials that can be used for analytical, industrial or therapeutic purposes (Feynman, R. P., In Miniaturization, ed. by H. D. Gilbert, Reinhold Publishing Corp., New York, 282-296 (1961); Drexler, K. E., Proc. Nat. Acad. Sci. (USA), 7: 5275-5278 (1981)). A particular aim is to construct individual objects and devices on the nanometer scale, utilizing the informational macromolecules, e.g., polypeptides and polynucleotides, of biological systems.
Nature provides many examples of elegant polypeptide or polynucleotide constructs on a nanometer scale. For example, the type of subcellular organelle known as a ribosome is a sophisticated machine constructed of polynucleotides and polypeptides. As is well known to the art, a ribosome functions in a machine-like manner in order to "read" the genetic sequence coded by a messenger ribonucleic acid (mRNA) strand and to generate the corresponding polypeptide.
Polypeptides play a variety of prominent functional roles in living cells, including enzymatic, regulatory and structural activities; hence, substantial effort has gone into the engineering of polypeptides (Leatherbarow, et al., Protein Eng., 1: 7-16 (1986); DeGrado, et al., Science, 243: 622-628 (1989); Anthony-Cahill, et al., Trends in Biochem. Sci., 14: 400-403 (1989)). However, less attention has been paid to the structural possibilities of nucleic acids The most stable form of naturally-occurring DNA is a linear double helical molecule (Watson, et al., Nature, 171: 737-738 (1953)), with limited potential for the construction of complex objects.
During the past several years, a number of investigations of the physical properties of deoxyribonucleic acid (DNA) branched junctions have been published (Kallenbach, et al. , Nature, 305; 829-831 (1983); Kallenbach, et al., J. Biomol. Str. and Dyns., 1: 158-168 (1983); Seeman, et al., Prog. Clin. & Biol. Res., 172A: 99-108 (1985); Wemmer, et al., Biochemistry, 24: 5745-5749 (1985); Marky, et al., Biopolymers, 26: 1621-1634 (1987); Churchill, et al., Proc. Nat. Acad. Sci. (USA), 85: 4653-4656 (1988); Chen, et al., Biochemistry, 27: 6032-6038 (1988); Cooper, et al., J. Mol Biol., 198: 711-719 (1987); Duckett, et al., Cell, 55: 79-89 (1988); Seeman, N. C., Electrophor., 10: 345-354 (1989); Cooper, et al., Proc. Nat. Acad. Sci., (USA) 86: 7336-7340 (1989); Murchie, et al., Nature, 341: 763-766 (1989)). Their susceptibility to resolving enzymes have also been reported (Duckett, et al., Cell, 55: 79-89 (1988); Evans, et al., J. Biol. Chem., 262: 14826-14836 (1987); Dickie, et al., J. Biol. Chem., 262: 14826-14836 (1987); Mueller, et al., Proc. Nat. Acad. Sci. (USA), 85: 9441-9445 (1988)). These studies have been stimulated by the role of branched DNA molecules as intermediates in the process of genetic recombination (Holliday, R., Genet. Res., 5: 282-304 (1964)).
In addition, the possibility of using branched DNA molecules to construct nanometer scale (also referred to herein as "nanoscale") objects has been explored In prior work a series of macrocycles (cyclic trimers, tetramers, etc.) was formed by oligomerizing 3-arm junctions (Ma, et al., Nucl. Acids Res., 14: 9745-9753 (1986)) or 4-arm junctions (Petrillo, et al., Biopolymers, 27: 1337-1352 (1988) containing a pair of complementary cohesive ends. The presence of numerous closed products from those ligations indicates a large degree of flexibility in the angles between arms (over long ligation times), regardless of how well-defined the structure of an individual junction may appear to be (Seeman, et al., Electrophor., 10: 345-354 (1989); Cooper, et al., Proc. Nat. Acad. Sci. (USA), 86: 7336-7340 (1989); Murchie, et al., Nature. 341: 763-766 (1989)).
Each of these studies has produced a closed object that may be described as 2-connected (Wells, A. F., Three-dimensional Nets and Polyhedra, John Wiley & Sons, New York, p. 3 (1977)). Although sharp kinks are introduced into the constructs by the presence of the junctions, the closed figures formed are essentially cyclic flexed variations on a linear theme.
The laboratory of the present inventors have made three dimensional DNA constructs where the DNA chains together formed a cube (Chen et al, Nature, 350: 631-633 (1991)), and copending patent application Ser. No. 07/639,684 filed Jan. 10, 1991, the entire contents of which are hereby incorporated by reference). This structure was formed from a series of preformed DNA constructs designed to specifically hybridize to each other.
Control over which arms are reactive in this previously described synthesis derives only from the presence or absence of 5' phosphates on particular exocyclic arms. Thus, only two logical stages of synthesis are possible if all strands are present throughout the synthesis: (i) initial phosphorylation of certain strands followed by ligation, and (ii) phosphorylation of the remaining strands in the intact molecule followed by a second ligation. Undesirable intermediate steps (denaturation and reconstitution) are necessary in the previous protocol, because it is not possible to purify side-products and failure products of the first reaction from the target product under native conditions.
Although it is possible to treat the ends of each branched component as individually accessible, the formation of closed geometrical objects entails two fundamentally different types of reactions, intermolecular additions and intramolecular cyclizations. Additions are favored usually by high concentrations of reactants, but cyclizations are favored by low concentrations. Thus, unless one wishes to run all the additions in a single step and all the cyclizations in a second one (sometimes possible, but unwise), greater control over multi-step synthesis must be obtained.
The synthesis of more complex structures from DNA will require greater control than that available in the synthesis of the cube-like object. Accordingly, the present inventors have developed a new methodology for the synthesis of DNA geometrical objects, in which each ligation may be separately performed.
Thus, it can be readily appreciated that the easily manipulated changes to form or modify a three dimensional structure of double stranded polynucleotides would be useful and a highly desirable advance over the current state of technology.